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| 1. |
Immerse the gel completing SDS-PAGE in transfer buffer and prepare a sheet of nitrocellulose (NC) membrane, 4 sheets of absorbent filter paper and 2 support
pads. |
| 2. |
Immerse nitrocellulose membrane soaked with distilled water in advance of the transfer buffer solution for 3 minutes. Also immerse absorbent filter paper in
the transfer buffer solution. |
| 3. |
Assemble transfer sandwich as following with immersed gel, membranes, filter papers, support pads.
Support pad | 2 sheets absorbent paper | gel | nc membrane | 2 sheets absorbent paper | support pad |
| 4. |
Put assembled sandwich in the transfer tank with the membrane facing towards the red-electrode. |
| 5. |
Transfer for 1 hour at 4℃ at 100 V. |
| 6. |
When transfer is complete, turn the power off and bring out the sandwich. |
| 7. |
Bring out nitrocellulose membrane and wash out several times with TBS-T buffer1. |
| 8. |
Stain the membrane by soaking in Ponceau S solution for 1-2 minutes. |
| 9. |
Confirm the transferred bands after washing with distilled water to distinguish protein bands well. |
| 10. |
React with blocking solution2 for 1 hours at room temperature. |
| 11. |
Treat with diluted primary antibody solution for 4 hours at room temperature. |
| 12. |
Wash out with TBS-T buffer three times for each time 10 minutes in duration. |
| 13. |
Add horseradish peroxidase(HRP)-labeled secondary antibody solution and incubate for about 1 hour at room temperature. |
| 14. |
Wash out with TBS-T buffer three times for 10 minutes each. |
| 15. |
Add fresh chemiluminescence reagent and incubate for 1 minute. |
| 16. |
Even after clearing surplus chemiluminescence reagent and drying out remaining wetness with paper towel, expose for 1 minute on plastic wrap for the film not
to be moistened. The time for chemiluminescence reagent incubation and file exposure may vary with the antibody used. |
